Cell-matrix biomechanical interactions play a critical role in both physiological and pathological processes such as embryonic tissue morphogenesis and wound repair. Despite general agreement that fibroblasts exert mechanical forces on the extracellular matrix (ECM) to promote organization of the collagen architecture, the underlying mechanisms of force transduction are not clearly understood. Based upon previous studies of in vivo corneal wound healing, we hypothesize that (1) mechanical forces are generated by a muscle-like contractile mechanism; and, (2) an isotropy in the ECM leads to a progressive alignment of this contractile machinery parallel to the axes of greatest mechanical resistance. We have recently developed a novel biophysical system that allows measurement of the forces generated by isolated corneal fibroblasts on a fibrillar collagen matrix and the direct correlation of ECM force vectors with specific cellular movements. Using this system, we have found that generation of force on the ECM correlates temporally with cellular contraction. While these pilot data are consistent with our original hypothesis, they do not yet definitively establish the role of contractile shortening in force generation, or the effect of anisotropy on cell alignment and tension generation. Thus, the overall goal of this Bioengineering Research Application is to develop a new, unique experimental system for directly and quantitatively correlating changes in protein organization with cellular force generation on fibrillar collagen matrix and to determine the effects of tissue anisotropy on the contractile response, using our existing biophysical system as a foundation. Our Specific Aims are to: (1) incorporate live-cell fluorescent imaging into our experimental model to allow simultaneous measurement of cell-induced matrix distortion and changes in the organization of contractile proteins; (2) use digital image analysis and finite element modeling to assess quantitatively the relationships between changes in contractile protein organization and cellular force generation; and (3) determine the effect of anisotropy in the ECM on the pattern of cellular force generation and contractile protein organization by inserting microneedles into the ECM in order to modulate matrix stiffness. This research should provide unique insights into the mechanical interactions between cells and ECM, and should serve as a critical foundation for future quantitative cell mechanics studies in this and other laboratories.